There is significant interpatient variation in drug metabolism. This is clinically important for drugs with narrow therapeutic indices such as the immunosuppressive agent cyclosporin A and anti-cancer agents including etoposide, teniposide, taxol and ifosphamide (Weber G. F. and Waxman, D. J. (1993) Biochem Pharm 45: 1685–1694; Relling, M. V. et al. (1994) Mol. Pharmacol. 45: 352–358). The clinical significance of wide variations in drug metabolism are realized as major effects on drug efficacy, drug toxicity and hence, therapeutic outcome. For these reasons, it is important to elucidate the factors that regulate variability in drug metabolism.
Genetic Polymorphisms in Drug-Metabolizing Enzymes
The number of man-made and natural environmental substances increases at an exponential rate, and many of these substances pose a health risk to exposed individuals. To protect the human population from the adverse effects of these agents, it is desirable to be able to identify the individuals within the population who are at a higher risk of developing the adverse effects. Molecular biology and molecular genetics have become essential tools in environmental toxicology. In fact, it is now feasible to approach experimentally one of the most challenging questions facing toxicologists today, namely, the identification of genes that contribute to an increased resistance (or sensitivity) to toxic environmental agents.
Susceptibility to chemical exposure, whether therapeutic, environmental, or occupational, is a well-documented phenomenon with a myriad of underlying causes, including age, health, gender, nutritional status, concominant therapy, and genetic factors. It is now well established that a major determinant of host-specific chemical susceptibility is the genetic variability observed in more than a dozen superfamilies of enzymes, collectively termed xenobiotic or drug-metabolizing enzymes. In addition to metabolizing the vast majority of chemicals to which humans are exposed on a daily basis, the members of these complex enzyme families also participate in many critical endogenous processes. Many drug-metabolizing enzymes have dual, often opposite, functions. For example, enzymes that detoxify lipid-soluble chemicals, by converting them to more readily excreted water-soluble metabolites, may also be capable of activating inert chemicals to highly reactive intermediates that interact with cellular macromolecules such as protein and DNA. Thus, for each chemical to which humans are exposed, there exist two potential competing pathways, one of metabolic activation and another of detoxification. Often these two pathways are exerted on the same compound, which becomes metabolically activated in order to be detoxified.
Subtle and gross differences in the genes encoding these enzymes have been identified and shown to result in marked variations in enzyme activity. These differences have been referred to as pharmacogenetic or, more broadly, ecogenetic polymorphisms (Motulsky, A. G. (1991) Pharmacogenetics 5:59). It has become apparent that each individual possess a distinct complement of drug-metabolizing enzyme activities and that it is the complex interplay of different enzyme variants that ultimately determines not only the fate of a chemical in any given individual, but also its potential toxicity. The development of relatively simple DNA-based tests, designed to identify specific gene alterations in these enzymes, can provide more accurate predictions of individual response to chemical exposure, thus expanding the field of preventive toxicology.
The vast majority of studies examining the relationship between genetic polymorphisms in drug-metabolizing enzymes and human health relate to the cytochrome P450 superfamily, the predominent phase I drug-metabolizing enzymes (Nebert, D. W. and Gonzales, F. J. (1987) Ann Rev Biochem 56:945; Nebert, D. W. and McKinnon, R. A. (1994) Prog. Liver Dis. 12:63). A member or members of the cytochrome P450 superfamily is involved in the metabolism of almost all chemicals to which humans are exposed. Found in animals, plants, fungi, yeast, and bacteria, these highly versatile enzymes also catalyze the oxidative metabolism of many critical endogenous substrates. The advent of cDNA cloning technology has resulted in rapid insights into the multiplicity of cytochrome P450 enzymes, with more than 500 distinct cytochrome P450 genes identified in all species (Nelson, D. R. et al., (1993) DNA Cell Biol 12:1; Nelson, D. B. et al., (1996) Pharmacogenetics 6:1). It has been estimated that humans may possess as many as 60 distinct P450 genes.
The genes in families CYP1, CYP2, and CYP3 of cytochrome P450 code for enzymes that are primarily responsible for the metabolism of most procarcinogens, promutagens, and drugs. The dual role of these enzymes in both detoxification and metabolic activation has prompted a vigilant survey of CYP genes for polymorphisms likely to result in variable enzymatic activities.
Cytochrome P450 (CYP) 3A Enzymes and Their Substrates
The cytochrome P450 proteins (CYPs) are a family of haem proteins resulting from expression of a gene super-family that currently contains over 500 members in species ranging from bacteria through to plants and animals. In humans, about 40 different CYPs are present and these play critical roles by catalyzing reactions in: (a) the metabolism of drugs, environmental pollutants and other xenobiotics; (b) the biosynthesis of steroid hormones; (c) the oxidation of unsaturated fatty acids to intercellular messengers; and (d) the stereo- and regio-specific metabolism of fat-soluble vitamins.
Quantitatively, the CYP3A family is the most abundantly expressed of the human CYP450s, representing on average 30% and 70% of total CYP450 in liver and intestine respectively (Shimada, T. et al. (1994) J. Pharmacol. Exp Therap 270:: 414–423). Four unique members of this gene family have been described. Sequences (cDNA) for CYP3A3 and CYP3A5 were cloned in the laboratory of P. S. Guzelian (Schuetz, J. D. et al. (1989) Arch Biochem Biophys 274: 355–365; Molowa D. T. et al. (1986) Proc. Natl. Acad Sci USA 83: 5311–5315). cDNAs for CYP3A7 and CYP3A4, the major CYP3A that is expressed in all humans have also been isolated (Beaune, P. H. et al. (1986) Proc. Natl Acad Sci USA 83: 8064–8068; Kitada, M. et al. (1987) J. Biol Chem 262: 13534–13537). One CYP3A5 pseudogene, CYP3A5P has been reported (Schuetz, J. D. et al (1995) Biochem. Biophys Act 1261: 161–165). A large number of drugs are mainly metabolised by the CYP3A subfamily (see TABLE 1). Therefore, maturational changes in CYP3A ontogeny may impact on the clinical pharmacokinetics of these drugs.
TABLE 1Important substrates for cytochrome P450 (CYP) 3ADRUGSAntihistaminesAntifungalsAnaesthesia-analgesicsAstemizoleKetoconazoleAlfentanilMizolastineMiconazoleFentanylTerfenadineImmunosuppressantsLidocaine (lignocaine)AntirefluxCyclosporinEthylmorphineCisapride(M1 formation)AntihypertensivesAnti-emeticCyclosporinAmlodipineOndansetron(M17 formation)FelodipineAnticonvulsantsTacrolimus (FK-506)IsradipineCarbamazepineChemotherapeuticsNicardipineCionazepamBusulfanNifedipineEthoxisumideDoxorubicinAnti-arrhythmicsZonisamideEtoposideVerapamilAnti-HIVTamoxifenQuinidineIndinavir(also CYP2D6)AntidepressentsRitonavirVinblastineImipramineSaquinavirVincristineNefazodoneAntimicrobialsBenzodiazepinesSertralineClindamycinAlprazolamMiscellaneousErythromycinDiazepam (minor)DextromethorphanRifampicin (rifampin)Midazolam (1-hydroxyformation)Midazolam (4-hydroxyformation)TemazepamTrizolamXENOBIOTICSAflatoxin B1Benzopyrene activationSterigmatocystinBenzphetamineHeterocyclic aminesENDOGENOUS SUBSTRATESAndrostanedioneEstradiolTestosterone(6β-hydroxylation)(2β-hydroxylation)Cortisol17 α-EthinylestradiolTestosterone(6β-hydroxylation)(6β-hydroxylation)Dehydroepiandro-ProgesteroneTestosteronesterone(6β-hydroxylation)(15β-hydroxylation)Dehydroepiandro-steronesulfateReferences for Table I:Michalets, E.L. (1998) Pharmacotherapy 18(1): 84–112.Kearns, G.L. (1995) Curr Opin Pediatr 7: 220–223
CYP3A4 is the most abundantly expressed CYP and accounts for approximately 30 to 40% of the total CYP content in human adult liver and 70% in the small intestine. CYP3A5 is 83% homologous to CYP3A4, is expressed at a lower level than CYP3A4 in the liver in some individuals—but may be the dominant CYP3A in up to 10% of Caucasians (K. Thummel, personal communication)—and is the main CYP3A isoform in the kidney and many extrahepatic tissues. CYP3A7 is the major CYP isoform detected in human embryonic, fetal and newborn liver, but is also detected in adult liver, although at a much lower level than CYP3A4.
Cytochrome P450 3A5
The substrate specificity of CYP3A5 appears to be similar to that of CYP3A4; however, some differences in catalytic properties have been found. In a reconstituted system, the formation rate of 1-hydroxy-midazolam is considerably higher for CYP3A5 than with CYP3A4. In contrast, the rate of formation of 4-hydroxy-midazolam with CYP3A4 and CYP3A5 is similar (Gorski, J. C. et al., (1994) Biochem Pharmacol 47(9): 1643–53). No CYP3A5 catalytic activity was found towards quinidine, 17α-ethinyl-estradiol and aflatoxins, all substrates of CYP3A4 (Wrighton, S. A. et al., (1990) Mol Pharmacol 38(2): 207–213). However, Gillam et al. did find considerable catalytic activity of CYP3A5 towards both erythromycin (about 6 times higher when compared with CYP3A4) and ethylmorphine (Gillam E. M. et al., (1995) Arch Biochem Biophys 317(2): 374–84). Interestingly, Gorski et al. found a much better correlation between midazolam hydroxylation and erythromycin N-demethylation when livers containing both CYP3A4 and CYP3A5 were excluded from analysis, a finding which supports the different isoform specificity for these drugs (Gorski, J. C. et al., (1994) Biochem Pharmacol 47(9): 1643–53).
A tabulation of the results of studies examining the substrate specificity of CYP3A isoforms 3A4, 3A5 and 3A7 is presented in TABLE 2.
TABLE 2Substrate specificity of the 3 cytochrome P450 (CYP) 3A isoforms and CYP3A-mediated metabolism during development in vitro.CYP isoformsexpressed in cellsCatalytic activity toward CYP3A substrates in human liver microsomesSubstrate3A43A53A7adult (%)Child (%)newborn (%)fetus (%)referencescommentsDrugsCarbamazepine++++++PresentCBZ-E 7, 15Age-dependant change informationmetabolites formed. CBZ-E(?)present in stillborn fetus ofmother receivingcarbamazepineCyclosporin (M1++++++ 3formation)Cyclosporin+++ND 33A5, less metabolites(M17 formation)formedDextromethorphan100 3016, 173MM formation (CYP3A)Diazepam100140 (3–12 mo)15 (<24 h <517Temazepam formation rate(minor)postpartum); 40–(CYP3A mediated)50 (1–7 dayspostpartum)Erythromycin+++ND/ 5, 6++++Ethylmorphine+++100100 6, 10Indinavir100 67 (6–11 y) 3214Lidocaine+++++?19Midazolam (1-+++++/− 1, 4hydroxyformation)Midazolam (4-+++++ 4, 20hydroxyformation)Nifedipine++++++100 44 18 3, 5, 13Paracetamol100 1021Also CYP2E1, sulphation(acetaminophen)and glucoronidationQuinidine+++ND 5Zonisamide++++++ 7Endogenous substratesAndrostanedione+++++++ 3, 22(6β-hydroxylation)Cortisol+++++− 5, 23DHEA(16α-++++++++ 1, 5, 8, 11hydroxylation)DHEA (16β-++++11hydroxylation)DHEA-S+++++++ 5, 7Progesterone (6β-+++++ 3, 9hydroxylation)Testosterone (2β-+++++++/<1 or 100 12–38 12 1, 2, 7, 13hydroxylation)++++Testosterone (6β-++++++100 30–4030–40 2–1012, 22, 24hydroxylation)Testosterone++++/− 3(15β-hydroxylation)a Relative to adult rate = 100%CBZ-E = CARBAMAZEPINE - 10, 11 eposide; DHEA = dehydroeplandrosterone; DHEA-S = dehydroepiandrosterone 3-sulphate; 3MM = methoxy-methorphan; ND = not detected; + to ++++ indicates increasing levels of expression; − indicates not expressed; ? indicated expression unknown.References1. Lacroix D. Sonnier M. Moncion A, et al. Expression CYP3A in the liver evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth. Eur J. Biochem 1997; 247: 625–34.2. Wrighton S A, Ring B J, Watkins P B, et al. Identification of a polymorphically expressed member of the human cytochrome P-450 III family. Mol Pharmacol 1989; 36(1): 97–105.3. Aoyama T. Yamano S. Waxman D J et al. Cytochrome P-450 hPCN3, a novel cytochrome P-450 III A gene product that is differentially expressed in adult human liver. cDNA and deduced amino acid sequence and distinct specificities of cDNA-expressed hPCN1 and hPCN3 for the metabolism of steriod hormones and cyclosporine. J Biol Chem 1989; 264 (18): 10388–95,4. Gorski J C, Hall S D, Jones D R, et al. Regioselective biotransformation of midazolam by members of the human cytochrome P450 3A (CYP3A) subfamly. Biochem Pharmacol 1994; 47 (9): 1643–53.5. Wrighton S A, Brian W R, Sari M A, et al Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3). Mol Pharmacol 1990; 38 (2): 207–13.6. Gillam E M, Guo Z. Ueng Y F, et al. Expression of cytochrome P450 3A5 in Escherichia coli: effects of 5′ modification, purification, spectral characterization, reconstitution conditions, and catalytic activities [published erratum appears in Arch Biochem Biophys 1995 Apr 20: 318 (2): 498]. Arch Biochem Biophys 1995: 317 (2): 374–84.7. Ohmori S. Nakasa H. Asanome K, et al. Differential catalytic properties in metabolism of endogenous and exogenous substrates among CYP3A enzymes expressed in COS-7 cells. Biochem Biophys Acta 1998; 1380 (3): 297–304.8. Kitada M. Kamataki T. Itahashi K. et al. P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, is the 16 alpha-hydroxylase of dehydroepiandrosterone 3-sulfate. J Biol Chem 1987; 262 (28): 13534–7).9. Schuetz J D, Beach D L, Guzelian P S. Selective expression of cytochrome P450 CYP3A mRNAs in embryonic and adult human live. Pharmacogentics 1994; 4: 11–20.10. Ladona M G, Spalding D F, Ekamn I, et al. Human fetal and adult liver metabolism of ethylmorphine. Relation to immunodected cytochrome P-450 PCN and interactions with important fetal corticosteroids. Biochem Pharmacol 1989: 38 (19): 3147–55.11. Cresteuk T, Beaune P, Kremers P, et al, Immunoquantification of epoxide hydrolase and cytochrome P-450 isozymes in fetal and adult human liver microsomes. Eur J Biochem 1985; 151 (2): 345–50.12. Shimada T. Yamazaki H, Mimura M. et al. Charcterization of microsomal cytochrome P450 enzymes involved in the oxidation of xenolbiotic chemicals in human fetal liver and adult lungs. Drug Metab Dispos 1996; 24 (5): 515–22.13. Chiba M. Nishime J A, Lin J F, et al. In vitro metabolism of indinavir in the human fetal liver microsome. Drug Metab Dispos 1997; 25 (10): 1219–22.14. Hakkola J. Pasanen M. Purkunen R, et al. Expression xenobiotic-metabolizing cytochrome P450 forms in human adult and fetal liver, Biochem Pharmacol 1994; 48 (1): 59–64.15. Piafsky K M, Rane A. Formaiton of carbamazepine epoxide in human fetal liver. Drug Metab Bispos 1978: 6 (4): 502–3.16. Jacqz.-Aigrain E. Funck-Brentano C, Cresteil T, CYP2D6- and CYP3A-dependent metabolism of dextromethorphan in humans. Pharmacogenetics 1993; 3: 197–204.17. Treluyer J M, Jacqz-Aigrain E. Alvarez F, et al. Expression of CYP2D6 in developing human liver. Eur J. Biochem 1991; 202 (2): 583–8.18. Treluyer J M, Gueret G, et al. Developmental expression of CYP2C and CYP2C-dependent activities in the human liver: in-vivo/in-vitro correlation and inducibility Pharmacogenetics 1997; 7 (6): 441–52.19. Bargetzi M J. Toshifumi A. Gonzalez F J, et al. Lidocaine metabolism in human liver muicrosomes by cytochrome P450 3A. Clin Pharmacol Ther 1989; 46 (5): 521–7.20. Li Y. Yokoi T. Sasaki M, et al. Perinatal expression and inducibility of human CYUP3A7 in C57BL/6N transgenic mice. Biochem Biophys Res Commun 1996; 228 (2): 312–7.21. Rollins D E, von Bahr C. Glaumann H, et al. Acetominophen potentially toxic metabolic formed by human fetal and adult liver microsomes and isolated fetal liver cells, Science 1979; 205: 1414–6.22. Macnpaa J. Pelkonen O, Cresteil T. et al. The role of cytochrome P450 3A (CYP3A isoform(s) in oxidative metabolism of testosterone and benzphetamine in human adult and fetal liver J. Steroid Biochem Mol Biol 1993: 44 (1): 61–7.23. Ohmori S. Fujiki N, Nakasa H, et al. Steroid hydroxylation by human fetal CYP3A7 and human NADPH-cytochrome P-450 reductase coexpressed in insect cells using baculovirus. Res Commun Mol Pathol Pharmacol 1998; 100 (1): 15–28.24. Kitada M. Kamataki T. Itahashi K, et al. Significance of cytochrome P-450 (P-450 HFLa) of human fetal livers in the steroid and drug oxidations. Biochem Pharmacol 1987; 36 (4): 453–6.
In summary, the specificity of CYP3A4 and 3A5 for the biotransformation of many substrates appears to be similar, although the extent and rate of metabolic conversion by the individual isoforms may be quite different for a given substrate. The discrepant results of in vitro studies probably reflect the sensitivity of CYP3A metabolic activities to incubation conditions.
Polymorphic Expression of CYP3A5
To a large extent, variation in drug metabolism is due to inter-individual differences in expression of CYP3A. It has been estimated that the metabolism of up to one-half of all commercially available drugs is catalyzed by a single dominant cytochrome P450 gene subfamily, the cytochromes P4503A (Cholerton, S. et al. (1992) Trends Pharmacol Sci. 13: 434–439). Thus, administration of a therapeutic dose of cyclosporin A (a CYP3A substrate) to a transplant patient with extremely low or high CYP3A is more likely to result in nephrotoxicity or graft organ rejection, respectively (Turgeon, K. et al. (1992) Clin. Pharmacol. and Therap. 52: 471–480). Similarly, administration of a standard dose of etoposide (a CYP3A substrate) to a cancer patient with low or high CYP3A is more likely to result in hematologic or gastrointestinal toxicities or treatment failure, respectively (Rodman, J. H. et al. (1994) J. Clin. Oncol. 12: 2390–97). Significant interpatient difference, exceeding 30-fold in some patient populations, has been demonstrated in both CYP3A content and its related catalytic activities among untreated human livers (Watkins, P. B. (1995) Hepatology 22: 994–996). Variation in CYP3A activities reflects, in part, the heterogeneous expression of the individual CYP3A family members.
Variation in Expression of Hepatic CYP3A5.
Polymorphic expression of CYP3A5 mRNA (Schuetz, J. D. (1989) Arch. Biochem Biophys. 274: 355–365) and CYP3A5 protein has been identified as one factor contributing to individual variation in total CYP3A expression and, thus CYP3A-mediated metabolism of drugs (Wrighton, S. A. et al. (1989) Mol Pharmacol 36: 97–105; Wrighton, S. A. et al. (1990) Mol. Pharmacol 38: 207–213). About 75% of the Caucasian population fail to express hepatic CYP3A5 mRNA or protein; these individuals are defined as CYP3A5 non-expressors. Within the 25% of the population defined as CYP3A5 hepatic expressors an additional level of variation in CYP3A5 expression exists. Among these individuals, the previously published content of CYP3A5 protein varies from 2 to 220 pmol CYP3A5/mg microsomal protein, and with respect to total CYP3A, ranges from <1% to as much as 100% (Aoyama, T. et al. (1989) J. Biol Chem 264: 10388–10395; Wrighton, S. A. et al. (1990) Mol Pharmacol 38: 207–213). The average content of CYP3A5 protein among expressors is reported as only approximately 30% that of CYP3A4, however, the factors controlling the expression of CYP3A5 are unknown.
CYP3A5 is the Predominant Extrahepatic Expressed Form of CYP3A.
In contrast to human liver, the majority of human kidneys express CYP3A5 protein (Schuetz, E. G. et al. (1992) Arch Biochem Biophys 294: 206–214), and a more recent report found that all human kidneys express CYP3A5 mRNA and protein but in a bimodal fashion −25% express high levels of CYP3A5; 75% express low levels of CYP3A5. (Haehner, B. O. et al. (1995) ISSX Proceedings 8:352 (Abstr). CYP3A5 mRNA is also reported to be expressed in human anterior pituitaries (Murray, G. I. (1995) FEBS Lett 364: 79–82). In contrast, the dominant hepatic form, CYP3A4, is only infrequently expressed in these extrahepatic tissues. These findings demonstrate that CYP3A5 is the predominant if not exclusive CYP3A family member expressed in most human extrahepatic tissues.
In order to understand the basis for the variable and polymorphic expression of CYP3A5 in the liver and the factors governing its expression in extrahepatic tissue, it is necessary and desired to identify the molecular mechanisms regulating its expression. The tissue-specific and developmental regulation of CYP3A5 suggests that the basis of the hepatic polymorphism lies in the regulation of its expression, rather than in the CYP3A5 structural gene. Hashimoto et al. have reported the gene structure of CYP3A4 and its transcriptional control (Hashimoto, H. et al. (1993) Eur. J. Biochem 218: 585–595). A unique element regulating hormonal responsiveness (dexamethasone) of CYP3A5 was reported by Schuetz et al. (Schuetz, J. D. et al. (1996) Mol Pharmac 49: 63–72). Nonetheless, little, if anything, is known about the factors controlling transcriptional activation.
Given the significant and important relevance of interpatient variation in drug metabolism to the success, choice, toxicity, and dosage of therapeutic drugs, the ability to monitor and predict drug metabolism or responsiveness is paramount. A knowledge of, and the ability to predict a patient's drug metabolism and appropriate therapeutic index, by determining the interindividual differences in expression of CYP3A, including CYP3A5 and CYP3A4, is therefore needed.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.